Use of lectin-magnetic separation (LMS) for detecting Toxoplasma gondii oocysts in environmental water samples

Proof-of-principle of lectin-magnetic separation (LMS) for isolating Toxoplasma oocysts (pre-treated with 0.5% acidified pepsin (AP)) from water for subsequent detection by microscopy or molecular methods has been shown. However, application of this technique in the routine water-analysis laboratory requires that the method is tested, modified, and optimized. The current study describes attempts to apply the LMS technique on supernatants from water samples previously analyzed for contamination with Cryptosporidium and Giardia using standard methods, and the supernatant following immunomagnetic separation (IMS) retained. Experiments on AP-treatment of Toxoplasma oocysts in situ in such samples demonstrated that overnight incubation at 37 °C was adequate, but excess AP had to be removed before continuing to LMS; neutralization in sodium hydroxide and a single wash step was found to be suitable. Mucilaginous material in post-IMS samples that had been stored at room temperature without washing, which was found to be probably an exudate from bacterial and fungal overgrowth, hampered the isolation of T. gondii oocysts by LMS beads. For detection, microscopy was successful only for clean samples, as debris occluded viewing in dirtier samples. Although qPCR was successful, for some samples non-specific inhibition occurred, as demonstrated by inhibition of an internal amplification control in the qPCR reaction. For some, but not all, samples this could be addressed by dilution. Finally, the optimized methodology was used for a pilot project in which 23 post-IMS water sample concentrates were analyzed. Of these, only 20 provided interpretable results (without qPCR inhibition) of which one sample was positive, and confirmed by sequencing of PCR product, indicating that Toxoplasma oocysts occur in Norwegian drinking water samples. In conclusion, we suggest that post-IMS samples may be suitable for analysis for Toxoplasma oocysts using LMS, only if freshly processed or washed before being refrigerated. In addition, application of AP treatment requires a neutralization step before proceeding to LMS. For detection, qPCR, rather than microscopy, is the most appropriate approach, although some inhibition may still occur, and therefore inclusion of an internal amplification control is important. Our study indicates that, despite some limitations, this approach would be appropriate for further large-scale analysis of samples of raw and treated drinking water.